This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Maintenance of genomic stability in the presence of genotoxic agents is a challenge to every cell, and failure to repair DNA lesions accurately may lead to aging, cancer and genetic diseases. The mutagenic potential of DNA lesions is largely determined by DNA polymerases specialized in translesion synthesis (TLS). TLS polymerases are intrinsically error-prone and capable of bypassing different types of damage with varying fidelity. Most TLS polymerases belong to a newly identified family, Y-family of DNA polymerases. Due to their mutagenic potential their activities must be tightly controlled. Sulfolobus solfataricus DNA polymerase IV (Dpo4) is a Y-family polymerase with low fidelity and a broad lesion-bypass spectrum. Previous studies show that this enzyme represents a good model system to study the mechanism of TLS and associated mutagenesis through structural analysis. We proposed that the active sites of Y-family polymerases remains especially open, even during catalysis, allowing them to accommodate the structurally distorted DNA lesions and mismatches of base pairs, and (2) that its key co-factor PCNA recruits the Y-polymerase to the replication fork during translesional DNA replication by different interactions to the normal replicative and translesion (Y) polymerases. In the structural study: 1) X-ray structural analysis of a series of complexes of Dpo4 and DNA fragments containing lesions and mismatches will be performed to visualize the detailed interactions between Dpo4 and DNA lesions at the atomic level and to develop a molecular model for lesion bypass and mutation, 2) Structure-guided mutagenesis on Dpo4 will be performed, followed by functional and structural characterization of the mutants to validate the model developed above. 3) X-ray structural analysis will be performed on PCNA/Dpo4 and PCNA/pol B (the main replicative polymerase in S. solfataricus) complexes to reveal specific interactions of PCNA with normal or translesion polymerases. Structural comparison of these two complexes should uncover differential PCNA binding interactions, which may control the recruitment of a specific polymerase to the replication fork during normal or translesion DNA synthesis.
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